The authors would like to thank Jeffrey Farrell, James Gagnon, and Jennifer Bergmann for comments on the manuscript. KWR was supported by the National Science Foundation Graduate Research Fellowship Program during the development of the FDAP assay. This work was supported by grants from the NIH to AFS and by grants from the German Research Foundation (Emmy Noether Program), the Max Planck Society, and the Human Frontier Science Program (Career Development Award) to PM.

1. Generating a Photoconvertible Fusion Construct and Injecting Dechorionated Zebrafish Embryos
<ol>
	<li>Generate a functional construct containing the protein of interest fused to a green-to-red photoconvertible protein (see Discussion), then use in vitro transcription to generate capped mRNA encoding the fusion protein as in M&#252;ller et al., 201219.</li>
	<li>Use pronase to remove the chorions from about 30 zebrafish embryos at the one-cell stage. Alternatively, manua...

The success of an FDAP experiment relies on the generation of a functional photoconvertible fusion protein. Tagging a protein can affect its biological activity and/or biophysical properties, including its localization, solubility, and stability36-41. Be prepared to test the activity of several different fusion constructs in order to find one that is active. We have found that changing the position of the photoconvertible protein relative to the protein of interest or using longer linkers (e.g., using...

The levels of proteins in cells and organisms are determined by their rates of production and clearance. Protein half-lives can range from minutes to days1-4. In many biological systems, the stabilization or clearance of key proteins has important effects on cellular activity. Modulation of intracellular protein stability is required for cell cycle progression5,6, developmental signaling7-9, apoptosis10, and normal function and maintenance of neurons11,12. Extracellular protein stability affects the distribution and availability of secreted proteins, such as morphogens13,14, within a tissue.
Over the last few decades, protein stability has mainly been assessed in cell culture using radioactive pulse-labeling or cycloheximide chase experiments15. In such pulse-chase experiments, cells are either transiently exposed to a &#8220;pulse&#8221; of radioactive amino acid precursors that are incorporated into newly synthesized proteins, or they are exposed to cycloheximide, which inhibits protein synthesis. Cultured cells are then collected at different time points, and either immunoprecipitation followed by autoradiography (in radioactive pulse-chase experiments) or western blotting (in cycloheximide experiments) is used to quantify the clearance of protein over time.
Conventional protein stability assays have several shortcomings. First, proteins in these assays are often not expressed in their endogenous environments, but rather in tissue culture and sometimes in cells from different species. For proteins whose stability is context-dependent, this approach is problematic. Second, it is not possible to follow protein clearance in individual cells or organisms over time, and the data from these assays reflects an average of different populations of cells at different time points. Since individual cells may have started with different amounts of protein, may have taken up the radioactive label or cycloheximide at different times, or may have different clearance kinetics, such aggregate data could be misleading. Finally, in the case of cycloheximide chase experiments, addition of the protein synthesis inhibitor may have unintended physiological effects that could artificially alter protein stability16-18. These shortcomings can be avoided by using Fluorescence Decay After Photoconversion (FDAP), a technique that utilizes photoconvertible proteins to measure protein clearance dynamically in living organisms19-25 (see Discussion for limitations of the FDAP technique).
Photoconvertible proteins are fluorescent proteins whose excitation and emission properties change after exposure to specific wavelengths of light26. One commonly used variant is Dendra2, a &#8220;green-to-red&#8221; photoconvertible protein that initially has excitation and emission properties similar to green fluorescent proteins, but after exposure to UV light&#8212;&#8220;photoconversion&#8221;&#8212;its excitation/emission properties become similar to those of red fluorescent proteins23,27. Importantly, new protein produced after photoconversion will not have the same excitation/emission properties as the photoconverted protein, allowing decoupling of production and clearance upon photoconversion and observation of only a pool of photoconverted protein. Tagging proteins of interest with photoconvertible proteins thus provides a convenient way to pulse-label proteins in intact, optically accessible living organisms.
In FDAP assays (Figure 1A), a protein of interest is tagged with a photoconvertible protein and expressed in a living organism (Figure 1B). The fusion protein is photoconverted, and the decrease in photoconverted signal over time is monitored by fluorescence microscopy (Figure 1C). The data is then fitted with an appropriate model to determine the half-life of the fusion protein (Figure 1D).
The FDAP assay described here was designed to determine the extracellular half-lives of secreted signaling proteins in zebrafish embryos during early embryogenesis19. However, this approach can be adapted to any transparent model organism that tolerates live imaging, and could be used to monitor the clearance of any taggable intracellular or extracellular protein. Variations of the technique described here have been performed in cultured cells20,23 and Drosophila22 and mouse21 embryos.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>PyFDAP (download from the following website: http://people.tuebingen.mpg.de/mueller-lab)</td>
			<td></td>
			<td></td>
			<td>Install and operate using the instructions provided on the PyFDAP website; PyFDAP is compatible with Linux, Mac, and Windows operating systems.</td>
		</tr>
		<tr>
			<td>mMessage mMachine Sp6 Transcription Kit (Life Technologies, AM1340)</td>
			<td></td>
			<td></td>
			<td>To generate capped mRNA for injection into embryos</td>
		</tr>
		<tr>
			<td>Alexa488-dextran conjugate, 3 kDa (Life Technologies, D34682)</td>
			<td></td>
			<td></td>
			<td>Co-inject with mRNA to create intracellular and extracellular masks</td>
		</tr>
		<tr>
			<td>6-well plastic dish (BD Falcon)</td>
			<td></td>
			<td></td>
			<td>Incubate embryos in agarose-coated wells until ready for mounting</td>
		</tr>
		<tr>
			<td>Embryo medium</td>
			<td></td>
			<td></td>
			<td>250 mg/l Instant Ocean salt, 1 mg/l methylene blue in reverse osmosis water adjusted to pH 7 with NaHCO3</td>
		</tr>
		<tr>
			<td>Protease from Streptomyces griseus (Sigma, P5147)</td>
			<td></td>
			<td></td>
			<td>Make a 5 mg/ml stock and use at 1 mg/ml to dechorionate embryos at the one-cell stage</td>
		</tr>
		<tr>
			<td>5 cm diameter glass petri dish</td>
			<td></td>
			<td></td>
			<td>For embryo dechorionation</td>
		</tr>
		<tr>
			<td>200 ml glass beaker</td>
			<td></td>
			<td></td>
			<td>For embryo dechorionation</td>
		</tr>
		<tr>
			<td>Microinjection apparatus</td>
			<td></td>
			<td></td>
			<td>For injection of mRNA and dye into embryos at the one-cell stage</td>
		</tr>
		<tr>
			<td>Stereomicroscope</td>
			<td></td>
			<td></td>
			<td>For injecting and mounting embryos</td>
		</tr>
		<tr>
			<td>1x Danieau&#39;s medium</td>
			<td></td>
			<td></td>
			<td>Dilute low melting point agarose and perform imaging in this medium; recipe: 0.2 mm filtered solution of 58 mM NaCl, 0.7 mM KCl, 0.4 mM MgSO4, 0.3 mM CaCl2, 5 mM HEPES pH 7.2</td>
		</tr>
		<tr>
			<td>UltraPure low melting point agarose (Invitrogen, 16520-100)</td>
			<td></td>
			<td></td>
			<td>For mounting embryos; use at a concentration of 1% in Danieau&#39;s medium: add 200 mg to 20 ml Danieau&#39;s medium, microwave until dissolved, then aliquot 1 ml into microcentrifuge tubes; aliquots can be stored at 4 &#176;C, re-melted at 70 &#176;C, and cooled to 40 - 42 &#176;C when ready to use</td>
		</tr>
		<tr>
			<td>Glass Pasteur pipette (Kimble Chase (via Fisher), 63A53WT)</td>
			<td></td>
			<td></td>
			<td>For mounting embryos; flame the tip to prevent jagged edges from injuring embryos</td>
		</tr>
		<tr>
			<td>Metal probe</td>
			<td></td>
			<td></td>
			<td>For positioning embryos during mounting</td>
		</tr>
		<tr>
			<td>Glass bottom dishes (MatTek, P35G-1.5-14-C)</td>
			<td></td>
			<td></td>
			<td>Use the appropriate cover glass thickness for your objective; part number listed here is for cover glass No. 1.5</td>
		</tr>
		<tr>
			<td>15 ml tube (BD Falcon) filled with ~5 ml embryo medium&#160;</td>
			<td></td>
			<td></td>
			<td>For rinsing residual agarose from the Pasteur pipette</td>
		</tr>
		<tr>
			<td>Inverted laser scanning confocal microscope</td>
			<td></td>
			<td></td>
			<td>A mercury arc lamp, 488 nm laser, 543 nm laser, and the appropriate filter sets are required</td>
		</tr>
		<tr>
			<td>Heated stage</td>
			<td></td>
			<td></td>
			<td>To maintain embryos at the optimal temperature of 28 &#176;C during the experiment</td>
		</tr>
		<tr>
			<td>Confocal software capable of time-lapse imaging</td>
			<td></td>
			<td></td>
			<td>Must be able to define multiple positions and automatically image them at defined intervals&#160;</td>
		</tr>
		<tr>
			<td>25x or 40x water objective</td>
			<td></td>
			<td></td>
			<td>Objective for imaging</td>
		</tr>
		<tr>
			<td>10x air objective</td>
			<td></td>
			<td></td>
			<td>Objective for photoconversion</td>
		</tr>
		<tr>
			<td>Immersion oil</td>
			<td></td>
			<td></td>
			<td>Immersion oil with the same refractive index as water</td>
		</tr>
	</tbody>
</table>

measuring protein stability in living zebrafish embryos using fluorescence decay after photoconversion (fdap)

Protein stability influences many aspects of biology, and measuring the clearance kinetics of proteins can provide important insights into biological systems. In FDAP experiments, the clearance of proteins within living organisms can be measured. A protein of interest is tagged with a photoconvertible fluorescent protein, expressed in vivo and photoconverted, and the decrease in the photoconverted signal over time is monitored. The data is then fitted with an appropriate clearance model to determine the protein half-life. Importantly, the clearance kinetics of protein populations in different compartments of the organism can be examined separately by applying compartmental masks. This approach has been used to determine the intra- and extracellular half-lives of secreted signaling proteins during zebrafish development. Here, we describe a protocol for FDAP experiments in zebrafish embryos. It should be possible to use FDAP to determine the clearance kinetics of any taggable protein in any optically accessible organism.

FDAP has been used to determine the half-lives of extracellular signaling proteins in zebrafish embryos19. One of these proteins, Squint, induces expression of mesendodermal genes during embryogenesis34. Squint-Dendra2 activates expression of mesendodermal genes at levels similar to untagged Squint, as demonstrated by qRT-PCR and in situ hybridization assays19. Embryos were co-injected with Alexa488-dextran and mRNA encoding Squint-Dendra2 and subjected to the FDAP assay. A decre...

Watch this Scientific Journal Video about Measuring Protein Stability in Living Zebrafish Embryos Using Fluorescence Decay After Photoconversion FDAP at JoVE.com

Measuring Protein Stability in Living Zebrafish Embryos Using Fluorescence Decay After Photoconversion FDAP

Protein levels in cells and tissues are often tightly regulated by the balance of protein production and clearance. Using Fluorescence Decay After Photoconversion (FDAP), the clearance kinetics of proteins can be experimentally measured in vivo.

Measuring Protein Stability in Living Zebrafish Embryos Using Fluorescence Decay After Photoconversion (FDAP)

Protein stability influences many aspects of biology, and measuring the clearance kinetics of proteins can provide important insights into biological ...

measuring-protein-stability-living-zebrafish-embryos-using

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JoVE Journal

Developmental Biology

11.1K visualizações.  Harvard University.  Os níveis de proteína em células e tecidos são muitas vezes fortemente regulada pelo equilíbrio da produção de proteínas e de apuramento. Usando Fluorescência Decay Após Fotoconversão (FDAP), a cinética de apuramento de proteínas pode ser medida experimentalmente in vivo. 